Herpes simplex virus (HSV) is a major cause of human disease and suffering. HSV is a widespread neurotropic DNA virus that initially causes an acute primary infection in the mucosal epithelium and then spreads to sensory ganglia where it establishes a latent lifelong infection. Approximately 54% of the US adult population is HSV-1 seropositive and 16% are HSV-2 seropositive. Primary infection may be asymptomatic but can result in severe disseminated disease including meningitis/encephalitis and pneumonia in the immunosuppressed, including neonates. Disseminated HSV infections exhibit high morbidity and mortality including neurologic sequelae in survivors, even if treated with acyclovir or other nucleoside analogs. Intermittent recurrences in latent disease can cause HSV keratitis, the most common cause of corneal blindness in the US, and recurrent genital herpes which is associated with significant morbidity. No vaccine exists and currently used antiviral medications like acyclovir target viral replication and are ineffective in eradicating latent virus. Our long-term goal is to develop novel CRISPR/SaCas9 based antiviral therapies for latent and acute HSV-1 and -2 infections and ultimately for infections with other clinically important members of the Herpesviridae family. In an attempt to develop new treatments for HSV, several studies have edited lytic HSV genomes, but no study has reported cleavage of latent/quiescent genomes. We have devised a multi-step in vitro screening approach and identified SaCas9 single guide RNAs (sgRNAs) that efficiently cleave quiescent HSV-1 genomes - the first demonstration that CRISPR-SaCas9 can target non-replicating HSV. We will expand our screen to HSV-2 and test the ability of CRISPR/SaCas9 to cleave the HSV-1 and -2 genome and abrogate latent and acute infections in mouse and human sensory neurons derived from pluripotent stem cells in vitro. We will then use an adeno-associated virus (AAV) based delivery system to test SaCas9/sgRNA combinations emerging from our screen for the treatment of HSV keratitis and neonatal encephalitis in animal models of HSV in vivo. In addition, we aim to understand the viral genomic factors modulating the efficiency of cleavage of individual SaCas9/sgRNA at HSV target sequences in the latent and lytic state. This project leverages a strong existing collaboration between two multi-disciplinary groups at Harvard University, which combines expertise in stem cell biology and CRISPR technology (Eggan Lab) and virology (Knipe Lab). If successful, the proposed study will establish the CRISPR/SaCas9 system as a novel prophylactic and therapeutic anti-viral strategy against HSV -1 and -2. This approach could be extended to other members of the Herpesviridae family as well as to other viruses without current effective treatment including RNA viruses. Our results will also pave the way for the generation of transgenic animals with constitutive expression of specific Cas9/guide RNAs conferring resistance against viral infections. This research is aligned with the NIH mission to develop novel biopharmaceutical approaches to the treatment of infection in humans.